Flow-Dependent Regulation of Kruppel-Like Factor 2 Is Mediated by MicroRNA-92a - PubMed (original) (raw)

. 2011 Aug 2;124(5):633-41.

doi: 10.1161/CIRCULATIONAHA.110.005108. Epub 2011 Jul 18.

Han Xiao, Andrés Laguna-Fernandez, Guadalupe Villarreal Jr, Kuei-Chun Wang, Greg G Geary, Yuzhi Zhang, Wei-Chi Wang, Hsien-Da Huang, Jing Zhou, Yi-Shuan Li, Shu Chien, Guillermo Garcia-Cardena, John Y-J Shyy

Affiliations

Flow-Dependent Regulation of Kruppel-Like Factor 2 Is Mediated by MicroRNA-92a

Wei Wu et al. Circulation. 2011.

Abstract

Background: Upregulated by atheroprotective flow, the transcription factor Krüppel-like factor 2 (KLF2) is crucial for maintaining endothelial function. MicroRNAs (miRNAs) are noncoding small RNAs that regulate gene expression at the posttranscriptional level. We examined the role of miRNAs, particularly miR-92a, in the atheroprotective flow-regulated KLF2.

Methods and results: Dicer knockdown increased the level of KLF2 mRNA in human umbilical vein endothelial cells, suggesting that KLF2 is regulated by miRNA. In silico analysis predicted that miR-92a could bind to the 3' untranslated region of KLF2 mRNA. Overexpression of miR-92a decreased the expression of KLF2 and the KLF2-regulated endothelial nitric oxide synthase and thrombomodulin at mRNA and protein levels. A complementary finding is that miR-92a inhibitor increased the mRNA and protein expression of KLF2, endothelial nitric oxide synthase, and thrombomodulin. Subsequent studies revealed that atheroprotective laminar flow downregulated the level of miR-92a precursor to induce KLF2, and the level of this flow-induced KLF2 was reduced by miR-92a precursor. Furthermore, miR-92a level was lower in human umbilical vein endothelial cells exposed to the atheroprotective pulsatile shear flow than under atheroprone oscillatory shear flow. Anti-Ago1/2 immunoprecipitation coupled with real-time polymerase chain reaction revealed that pulsatile shear flow decreased the functional targeting of miR-92a precursor/KLF2 mRNA in human umbilical vein endothelial cells. Consistent with these findings, mouse carotid arteries receiving miR-92a precursor exhibited impaired vasodilatory response to flow.

Conclusions: Atheroprotective flow patterns decrease the level of miR-92a, which in turn increases KLF2 expression to maintain endothelial homeostasis.

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Figures

Figure 1

Figure 1. miRNAs are involved in the regulation of KLF2 mRNA

(A) HUVECs were subjected to laminar flow (12 dyn/cm2) for 6 hr. After the addition of DRB (2 µg/ml), then the cells were continuously exposed to laminar flow or static conditions for additional 2 and 4 hr. The levels of KLF2 and GAPDH mRNAs were measured by qRT-PCR, and the KLF2/GAPDH mRNA ratio is plotted as a percentage of that in the untreated static cells. (B,C) HUVECs were transfected with 20 nM Dicer siRNA or control RNA for 48 hr. qRT-PCR (B) and Western blot analysis (C) were performed to detect the mRNA levels of KLF2 and eNOS and protein levels of KLF2, eNOS, and TM, respectively. Histone H1 served as the internal control of nuclear extracts. The bar graphs are mean±SD from 3 independent experiments. * p<0.05 between Dicer siRNA and control RNA, analyzed by ANOVA followed by Dunnett’s test (A) or Student t test (B,C). (D) The miRNA binding sites in the KLF2 3’UTR are predicted by bioinformatics algorithms. (E) The seed region of miR-92a and its target sequences at the KLF2 3’UTR of several mammalian species: Homo sapiens, Pan troglodytes, Mus Musculus, and Rattus Norvegicus

Figure 2

Figure 2. miR-92a targets KLF2 mRNA and decreases KLF2 translation

HUVECs were transfected with 20 nM miR-92a precursor (pre-92a) or control RNA. At 48 hr, the levels of miR-92a relative to U6 RNA and KLF2, eNOS, and TM mRNA as ratios to GAPDH were assessed by qRT-PCR. In (B) and (C), HUVECs were transfected with 20 nM miR-92a inhibitor (anti-92a) or control RNA. The mRNA levels of KLF2, eNOS, and TM as ratios to GAPDH were assessed by qRT-PCR. T h e protein levels of KLF2, Histone H1 and eNOS were determined by Western blotting. (D) HEK293 cells were transfected with the wild-type FLAGKLF2 (WT), FLAG-KLF2 (mut) (miR-92a binding site mutation), or Flag-KLF2 (Δ) (miR-92a 24 binding site deletion) together with 20 nM pre-92a or control RNA for 48 hr. The cells were then lysed, and the level of exogenously expressed FLAG-KLF2 fusion proteins was detected by Western blot analysis with anti-FLAG. Shown in the bottom panel is the densitometry analysis of the protein amount normalized to that in the control RNA-transfected cells. (E) HEK293 cells were transfected with the wild-type Luc-KLF2-3’UTR(WT) or Luc-KLF2-3’UTR(mut) together with 20 nM pre-92a or control RNA and CMV-β-gal. The luciferase activity was normalized to that of β-gal. The data represent mean±SD from 3 independent experiments. * p<0.05 between cells transfected with pre-92a and control RNA.

Figure 3

Figure 3. Shear stress-induction of KLF2 is mediated through miR-92a

(A) HUVECs were exposed to laminar flow for 4, 8 or 16 hr. qRT-PCR was performed to detect the level of miR-92a, which was normalized to that of U6 RNA. * p<0.05 compared with static controls (time 0), analyzed by one-way ANOVA followed by Dunnett’s test. (B–F) HUVECs were transfected with 20 nM control RNA or pre-92a for 48 hr and then exposed to laminar flow for 8 hr. (B) KLF2 mRNA level was detected by qRT-PCR and (C) protein level was assessed by Western blot analysis. (D,E) eNOS and TM mRNA levels were detected by qRT-PCR and (F) protein level was assessed by Western blot analysis, and the results of statistical analyses are shown in the right. The data represent mean±SD from 3 independent experiments. * p<0.05 between the indicated groups, analyzed by two-way ANOVA followed by the Bonferroni posthoc test.

Figure 4

Figure 4. PS down-regulates, but OS up-regulates, miR-92a expression in ECs

(A) HUVECs were exposed to PS (12±4 dyn/cm2) or OS (0±4 dyn/cm2) for 8 hr. qRT-PCR was performed to detect the level of miR-92a, which was normalized to that of U6 RNA. (B) The expression of miR-92a in ECs exposed to PS or OS flow assessed by miRNA microarray. (C,D) BAECs were transfected with Luc-2×miR92 reporter or control plasmid for 24 hr and then exposed to PS or OS flow for 12 hr. The luciferase activity was measured and normalized to β-gal activity. (E, F) BAECs were transfected with Luc-KLF2(WT) or Luc-KLF2(mut) for 24 hr and then exposed to PS or OS flow for 12 hr. The luciferase activity was measured and normalized to β-gal activity. The data represent mean±SD from 3 independent experiments. * p<0.05 between the 2 groups being compared by Student t test (A,B) or two-way ANOVA (C–F) followed by the Bonferroni posthoc test.

Figure 5

Figure 5. miRISC regulates miR-92a

HUVECs were exposed to PS (A,C) or OS (B,D) for 8 hr. The Ago1- or Ago2-associated miRNAs and mRNAs were enriched by IP with the use of anti-Ago1 (A,B) or anti-Ago2 (C,D). mRNA levels of miR-92a and KLF2 were detected by qRT-PCR and normalized to those of Ago1 or Ago2 protein. The data represent mean±SD from 3 independent experiments. * p<0.05 for PS or OS vs. static control, as analyzed by Student t test.

Figure 6

Figure 6. miR-92a regulates endothelial function in vitro and ex vivo

(A) HUVECs were transfected with control RNA or anti-92a. After 48 hr, the NO bioavailability was detected by fluorometric assay and expressed as nmol/106 cells. In (B), HUVECs were transfected with pre-92a and infected with Ad-KLF2-3’UTR or Ad-null for 48 hr. The level of KLF2 and eNOS mRNA was assessed by qRT-PCR and NO bioavailability was measured. (C) pre-92a or control RNA was administered to the carotid arteries of C57BL6 mice by pluronic gel F-127. Five days later, the arteries were isolated. The expression levels of miR-92a, KLF2 and eNOS in the isolated vessels (n=6) were assessed by qRT-PCR. miR-92a level was normalized to that of U6 RNA, whereas KLF2 and eNOS levels were normalized to that of GAPDH. The data represent mean±SD. (D) The flow-induced vasodilation ex vivo was measured by use of the SoftEdge Acquisiton Subsystem in the presence or absence of L-NAME (1 µM). The dilation ability is defined as the percentage of the diameter change of the flow-induced dilation compared to the diameter change of the PE-induced constriction. The bars represent mean±SEM. p<0.05 between the 2 groups being compared by Student t test (A,C) or two-way ANOVA followed by the Bonferroni posthoc test (B,D).

Figure 7

Figure 7. Shear stress regulation of KLF2

The diagram shows the regulatory circuitry of the responses of transcription factors and miRNAs to atheroprotective shear flow. The circled numerals refer to the Table numbers. Shear stress with a forward direction regulates the expressions of KLF2 and miR-92a through several TFs (in Supplemental Tables 2 and 3, respectively). Serving as a transcription factor, KLF2 transactivates the expression of downstream genes such as eNOS and TM. In addition, KLF2 may bind to the promoter region of some miRNAs, including miR-126, to upregulate their transcription directly (Supplemental Table 4). In turn, the network of KLF2 and miRNAs regulates the expression of factors that control anti-inflammatory, anti-thrombotic, anti-proliferative, anti-angiogenic, anti-oxidant, and anti-fibrotic effects to maintain EC functions. The methods for computational analysis are described in the supplements.

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